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The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a human receptor tyrosine kinase, KDR, which is expressed on human endothelial cells. This receptor is activated by VEGF and mediates a mitogenic signal. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding human KDR, a DNA fragment encoding the intracellular portion of KDR, a DNA fragment encoding the extracellular portion of KDR with or without a membrane anchor sequence, substantially purified forms of associated human KDR, and human mutant forms of KDR.
Vascular endothelial cells form a luminal non-thrombogenic monolayer throughout the vascular system. Mitogens promote embryonic vascular development, growth, repair and angiogenesis in these cells. Angiogenesis involves the proteolytic degradation of the basement membrane on which endothelial cells reside followed by the subsequent chemotactic migration and mitosis of these cells to support sustained growth of a new capillary shoot. One class of mitogens selective for vascular endothelial cells include vascular endothelial growth factor (referred to as VEGF or VEGF-A) and the homologues placenta growth factor (PlGF), VEGF-B and VEGF-C.
Human VEGF exists as a glycosylated homodimer in one of four mature processed forms containing 206, 189 (see U.S. Pat. No. 5,240,848), 165 (see U.S. Pat. No. 5,332,671), and 121 (U.S. Pat. No. 5,332,671) amino acids, the most prevalent being the 165 amino acid form. The 206 amino acid and 189 amino acid forms of human VEGF each contain a highly basic 24-amino acid insert that promotes tight binding to heparin, and presumably, heparin proteoglycans on cellular surfaces and within extracellular matrices (Ferrara et al., 1991, J. Cell. Biochem. 47:211-218).
Human PlGF is also a glycosylated homodimer which shares 46% homology with VEGF at the protein level. Differential splicing of human PlGF mRNA leads to either a 170 or 149 amino acid residue precursor, which are proteolytically processed to mature forms of 152 or 131 amino acid residues in length, respectively (Maglione et al., 1993, Oncogene 8: 925-931; Bayne and Thomas, 1992, EPO Publication No. 0 506 477 A1; Hauser and Weich, 1993, Growth Factors 9: 259-268).
VEGF-B has been isolated and characterized (Grimmond et al., 1996, Genome Research 6: 124-131; Olofsson et al., 1996, Proc. Natl. Acad. Sci. USA 93: 2576-2581). The full-length human cDNAs encode 188 and 207 amino acid residue precursors wherein the NH2 terminal portions are proteolytically processed to mature forms 167 and 186 amino acid residues in length. Human VEGF-B expression was found predominantly in heart and skeletal muscle as a disulfide-linked homodimer. However, human VEGF-B may also form a heterodimer with VEGF (id. @ 2580).
VEGF-C has also been isolated and characterized (Joukov et al., 1996, EMBO J. 15: 290-298). A cDNA encoding VEGF-C was obtained from a human prostatic adenocarcinoma cell line. A 32 kDa precursor protein is proteolytically processed to generate the mature 23 kDa form, which binds the receptor tyrosine kinase, Flt-4.
VEGF and its homologues impart activity by binding to vascular endothelial cell plasma membrane-spanning tyrosine kinase receptors which then activate an intracellular mitogenic signal. The KDR receptor family is the major tyrosine kinase receptor which transduces the mitogenic signal initiated by VEGF.
Shibuya et al. (1990, Oncogene 5: 519-524) disclose a human receptor type tyrosine kinase gene flt, which comprises a 4.2 Kb open reading frame encoding a 1338 amino acid protein which comprises a glycosylated extracelluar domain, membrane spanning region and predicted tyrosine kinase domain.
Pajusola et al. (1992, Cancer Res. 52: 5738-5743) disclose a human receptor type tyrosine kinase gene which, as noted above, binds human VEGF-C.
Vascular endothelial growth factor (VEGF) binds the high affinity membrane-spanning tyrosine kinase receptors KDR and Flt-1. Cell culture and gene knockout experiments indicate that each receptor contributes to different aspects of angiogenesis. KDR mediates the mitogenic function of VEGF whereas Flt-1 appears to modulate non-mitogenic functions such as those associated with cellular adhesion. Inhibiting KDR thus significantly diminishes the level of mitogenic VEGF activity.
Vascular growth in the retina leads to visual degeneration culminating in blindness. VEGF accounts for most of the angiogenic activity produced in or near the retina in diabetic retinopathy. Ocular VEGF mRNA and protein are elevated by conditions such as retinal vein occlusion in primates and decreased pO2 levels in mice that lead to neovascularization. Intraocular injections of either anti-VEGF mono-clonal antibodies or VEGF receptor immunofusions inhibit ocular neovascularization in rodent and primate models. Regardless of the cause of induction of VEGF in human diabetic retinopathy, inhibition of ocular VEGF is useful in treating the disease.
Expression of VEGF is also significantly increased in hypoxic regions of animal and human tumors adjacent to areas of necrosis. Monoclonal and polyclonal anti-VEGF antibodies inhibit the growth of human tumors in nude mice. Although these same tumor cells continue to express VEGF in culture, the antibodies do not diminish their mitotic rate of most, if not all, tumor cells derived from cells other than vascular endothelial cells themselves. Thus tumor-derived VEGF does not function as an autocrine mitogenic factor for most tumors. Therefore, VEGF contributes to tumor growth in vivo by promoting angiogenesis through its paracrine vascular endothelial cell chemotactic and mitogenic activities. These monoclonal antibodies also inhibit the growth of typically less well vascularized human colon cancers in athymic mice and decrease the number of tumors arising from inoculated cells. Viral expression of a VEGF-binding construct of Flk-1, the mouse KDR receptor homologue, truncated to eliminate the cytoplasmic tyrosine kinase domains but retaining a membrane anchor, virtually abolishes the growth of a transplantable glioblastoma in mice presumably by the dominant negative mechanism of heterodimer formation with membrane-spanning endothelial cell VEGF receptors. Embryonic stem cells, which normally grow as solid tumors in nude mice, do not produce detectable tumors if both VEGF alleles are knocked out. Taken together, these data indicate the role of VEGF in the growth of solid tumors. KDR and Flt-1 are implicated in pathological neoangiogenesis, and inhibitors of these receptors are useful in the treatment of diseases in which neoangiogenesis is part of the overall pathology, e.g., diabetic retinal vascularization, various forms of cancer as well as forms of inflammation such as rheumatoid arthritis, psoriasis, contact dermatitis and hypersensitivity reaction.
Terman et al. (1991, Oncogene 6: 1677-1683; 1992, Biochem. Biophys. Res. Commun. 187: 1579-1586) disclose a full-length cDNA encoding a form of KDR. However, the Terman et al. disclosures do not identify a novel, optimal nucleic acid fragment encoding the human form of the receptor type tyrosine kinase gene, KDR. It will be advantageous to identify and isolate a human cDNA sequence encoding an optimized form of human KDR. A nucleic acid molecule expressing the human KDR protein will be useful in screening for compounds acting as a modulator of the protein kinase domain of this protein. Such a compound or compounds will be useful in modulating the mitogenic signal of VEGF and VEGF-related proteins on vascular endothelial cells. The KDR nucleic acid sequence may be also useful for gene therapy encoding a portion of the KDR protein that would contain functional ligand binding and membrane anchoring moieties but not tyrosine kinase activity. Either all or a portion of the KDR protein is also useful to screen for VEGF antagonists. The KDR nucleic acid sequence can be transfected into cells for analysis of function in the absence of Flt-1. The KDR protein is also useful for x-ray structure analysis in the presence or absence of ligand and/or inhibitors. The present invention addresses and meets these needs by disclosing an isolated nucleic acid fragment which expresses a form of human KDR which is shown by computer modeling to be predictive of higher activity and functionality than the previously disclosed KDR.
The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a novel human receptor type tyrosine kinase gene, KDR. This specification discloses a novel, optimized DNA molecule which encodes, KDR, a receptor tyrosine kinase expressed on human endothelial cells.
The present invention also relates to biologically active fragments or mutants of SEQ ID NO:1 which encodes mRNA expressing a novel human receptor type tyrosine kinase gene, KDR. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment comprising at least an intracellular or extracelluar kinase domain similar to that of the human KDR protein as set forth in SEQ ID NO:2. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for KDR function.
The isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
The present invention also relates to subcellular membrane fractions of the recombinant host cells (both prokaryotic and eukaryotic as well as both stably and transiently transformed cells) comprising the nucleic acids of the present invention. These subcellular membrane fractions will comprise either wild-type or human mutant forms of KDR at levels substantially above wild-type levels and hence will be useful in various assays described throughout this specification.
A preferred aspect of the present invention is disclosed in FIG. 1A and FIG. 1B and SEQ ID NO:1, a human cDNA encoding a novel receptor type tyrosine kinase gene, KDR.
The present invention also relates to a substantially purified form of the receptor type tyrosine kinase gene, KDR which is disclosed in FIG. 2 and as set forth in SEQ ID NO:2.
The present invention also relates to biologically active fragments and/or mutants of the KDR protein as initially set forth as SEQ ID NO:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for KDR function.
A preferred aspect of the present invention is disclosed in FIG. 2 and is set forth as SEQ ID NO:2, the amino acid sequence of the novel receptor type tyrosine kinase gene, KDR.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the human form of KDR disclosed herein, or a biologically active fragment thereof.
The present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type human KDR activity. A preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase (GST)-KDR fusion constructs. These fusion constructs include, but are not limited to, either the intracellular tyrosine kinase domain of human KDR as an in-frame fusion at the carboxy terminus of the GST gene or the extracellular ligand binding domain fused to an immunoglobulin gene by methods known to one of ordinary skill in the art. Soluble recombinant GST-kinase domain fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (pAcG2T, Pharmingen).
The present invention also relates to isolated nucleic acid molecules which encode human KDR protein fragments comprising a portion of the intracellular KDR domain. The protein fragments are useful in assays to identify compounds which modulate wild-type human KDR activity. A preferred aspect of this portion of the invention includes, but is not limited to, a nucleic acid construction which encodes the intracellular portion of human KDR, from about amino acid 780-795 to about amino acid 1175-1386.
Therefore, the present invention relates to isolated nucleic acid molecules which encode human KDR protein fragments comprising a portion of the extracellular KDR domain. These isolated nucleic acid proteins may or may not include nucleotide sequences which also encode the transmembrane domain of human KDR. These KDR extracellular and/or KDR extracellular-transmembrane domain protein fragments will be useful in screening for compounds which inhibit VEGF binding as well as utilizing these isolated nucleic acids as gene therapy vehicles to inhibit VEGF-mediated mitogenic activity. Expression of either a soluble version of KDR (extracellular) or membrane bound form (extracellular-transmembrane) will inhibit in vivo VEGF/KDR mediated angiogenesis.
Therefore, the present invention relates to methods of expressing the receptor type tyrosine kinase gene, KDR, and biological equivalents disclosed herein, assays employing these receptor type tyrosine kinase genes, cells expressing these receptor type tyrosine kinase genes, and compounds identified through the use of these receptor type tyrosine kinase genes and expressed human KDR protein, including one or more modulators of the human KDR-dependent kinase either through direct contact with the kinase domain of human KDR or a compound which prevents binding of VEGF to human KDR, or appropriate dimerization of the KDR receptor antagonizing transduction of the normal intracellular signals associated with VEGF-induced angiogenesis.
The present invention also relates to gene therapy applications, especially for nucleic acid fragments which encode soluble extracelluar protein fragments of human KDR. It is disclosed herein that such methods will be useful especially in the treatment of various tumors as well as diabetic retinopathy.
It is an object of the present invention to provide an isolated nucleic acid molecule which encodes a novel form of human KDR, or human KDR fragments and KDR mutants which are derivatives of SEQ ID NO:2 and preferably retain Val at position 848, and especially preferable is retention of Val at position 848, Glu at position 498, Ala at position 772, Arg at position 787, Lys at position 835 and Ser at position 1347. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for KDR function.
It is a further object of the present invention to provide the human KDR proteins or protein fragments encoded by the nucleic acid molecules referred to in the preceding paragraph.
It is also an object of the present invention to provide biologically active fragments or mutants of human KDR which comprise an intracellular kinase domain similar to that of the human KDR protein as set forth in SEQ ID NO:2, preferably retaining Val at position 848, and especially preferable is retention of Val at position 848, Glu at position 498, Ala at position 772, Arg at position 787, Lys at position 835 and Ser at position 1347.
It is a further object of the present invention to provide recombinant vectors and recombinant host cells which comprise a nucleic acid sequence encoding human KDR or a biological equivalent thereof.
It is an object of the present invention to provide a substantially purified form of the receptor type tyrosine kinase gene, KDR, as set forth in SEQ ID NO:2.
It is an object of the present invention to provide for biologically active fragments and/or mutants of the KDR protein, such as set forth in SEQ ID NO:2, including but not necessarily limited to ammo acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
It is also an object of the present invention to provide for KDR-based in-frame fusion constructions, methods of expressing the receptor type tyrosine kinase gene, KDR, and biological equivalents disclosed herein, related assays, recombinant cells expressing these receptor type tyrosine kinase genes, and agonistic and/or antagonistic compounds identified through the use of these receptor type tyrosine kinase genes and expressed human KDR protein.
As used herein, xe2x80x9cVEGFxe2x80x9d or xe2x80x9cVEFG-Axe2x80x9d refers to vascular endothelial growth factor.
As used herein, xe2x80x9cKDRxe2x80x9d or xe2x80x9cFLK-1xe2x80x9d refers to kinase insert domain-containing receptor.
As used herein, xe2x80x9cFLT-1xe2x80x9d refers to fins-like tyrosine kinase receptor.
As used herein, the term xe2x80x9cmammalian hostxe2x80x9d refers to any mammal, including a human being.